Abstract The goal of this proposal is to investigate mechanisms for pyroptosis-dependent and novel pyroptosis- independent export of proinflammatory cytokines mediated by caspases, secretory autophagy proteins, and gasdermin D (GSDMD), a recently identified effector molecule for pyroptosis. The inflammasomes are crucial innate immune signaling platforms implicated in immune defense against infections and autoimmune/ autoinflammatory disorders such as inflammatory bowel disease, multiple sclerosis, and Alzheimer?s disease. Activation of the inflammasome signaling pathways leads to the maturation and secretion of cytokines such as IL-1b and IL-18, and an inflammatory form of programmed cell death in certain cell types called pyroptosis. GSDMD assembles membrane pores upon cleavage by inflammatory caspases-1, 4, 5 and 11 and caspase-8. In macrophages, such pores allow the release of mature cytokines upon cytolysis. Intriguingly, pyroptosis- and membrane pore-independent function of GSDMD during secretion of IL-1b upon inflammasome activation has been demonstrated for neutrophils, epithelial cells and T cells (Projects 1, 2, and 4), in which GSDMD is recruited by secretory autophagy proteins to facilitate a novel non-lytic form of cytokine release. Specific recognition of GSDMD by caspases or autophagy proteins thus underlie GSDMD function in lytic or non-lytic cytokine release. We hypothesize that inflammatory caspases recognize GSDMD through distinct protein-protein interfaces during lytic cytokine release. By contrast, GSDMD is recruited by autophagy machinery to facilitate novel non-lytic cytokine secretion independent of membrane pore formation. We will focus on the following specific aims in a collaborative Program Project to test the above hypothesis. Aim 1. Define the mechanisms of protease- dependent activation of GSDMD in lytic release of cytokines. We propose to elucidate the mechanisms of GSDMD recognition by caspases and serine proteases in collaboration with Projects 1, 2 and 4. Discovery from our proposal may facilitate investigation into specific caspase inhibitors based on distinct enzyme-substrate interfaces, which may target pyroptosis, apoptosis and other inflammatory signaling pathways involving caspases. Aim 2. Define the mechanisms of GSDMD-guided non-lytic secretion of cytokines. We propose to characterize the recruitment of IL-1b and GSDMD by chaperones and autophagy proteins. The roles of these interaction in non-lytic cytokine release will be probed in collaboration with Projects 2 and 4. The success of this project will reveal the molecular mechanisms for the lytic or non-lytic secretion of proinflammatory cytokines mediated by GSDMD. This proposal draws on the strength of the other three Projects to facilitate and validate our structural studies. Structural insights from our studies will in turn inform experimental design and data interpretation for the other Projects. As such, outcomes from the four Projects benefit each other as an interdependent and integrated Program.